Investigation of the Antibacterial and Antibiofilm Activity of Selenium Nanoparticles against Vibrio cholerae as a Potent Therapeutics

Vibrio cholerae is a major cause of severe diarrhea, which is ecologically flexible, and remains as a major cause of death, especially in developing countries. Consecutive emergence of antibiotic-resistant strains is considered to be as one of the major concerns of the World Health Organization (WHO). Nanoparticles as a new nonantibiotic therapeutic strategy have been widely used in recent years to treat bacterial infections. The present study aimed to investigate the antibacterial and antibiofilm effect of selenium nanoparticles (SeNPs) in vitro against V. cholerae O1 ATCC 14035 strain. SeNPs were prepared and characterized using ultraviolet-visible (UV-Vis) spectroscopy, DLS (dynamic light scattering), zeta potential measurement, and Fourier transform infrared (FTIR) analysis. The concentration of SeNPs was calculated by ICP (inductively coupled plasma) method. Also, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to assess the cytotoxic effect of SeNPs on Caco-2 cells. Antibacterial and antibiofilm activity of SeNPs was determined by broth microdilution and crystal violet assays, respectively. The average particle size of SeNPs was 71.1 nm with zeta potential −32.2 mV. The SEM images supported the uniform spherical morphology of the prepared nanoparticles. The antibiofilm effect of SeNPs was evident at concentrations of 50–200 μg/mL. This study results provided evidence that SeNPs are safe as an antibacterial and antibiofilm agent against V. cholerae O1 ATCC 14035 strain.


Introduction
Recently, nanotechnology is designed and applied in the pharmaceutical industry. Nanomaterials have extensive applications in the field of biotechnology, medicine, and chemistry due to their small particle size, targeted effects, fewer side effects, solubility, and pharmacokinetics; their biodistribution is also easy to administer, offering a market advantage to their developers [1].
Cholera epidemics are considered as an important public health concern in developing countries. So far, Vibrio cholerae has caused seven cholera pandemics in the world. According to the World Health Organization (WHO), 1.3 to 4.0 million new cases of cholera are diagnosed each year, resulting in 21000 to 143000 deaths per year worldwide [2]. Antibiotic treatment is an adjunct therapy used for acute diarrhea in cholera patients to reduce the duration and severity of diarrhea; it is also a beneficial measure that could be taken to control cholera epidemics. However, WHO does not recommend the indiscriminate use of antibiotics because overuse of antibiotics contributes to the emergence of antimicrobial resistance and multiple antibiotic resistance (MAR).
is antimicrobial resistance makes bacterial infections more difficult to treat and increases fatality rates during cholera outbreaks. However, many countries have reported the emergence of toxigenic V. cholerae strains resistant to frequently used antimicrobial agents [3]. erefore, the development of new strategies to combat V. cholerae infections is a necessity, and the solution may lie in nanotechnology [4]. Among nanoparticles, selenium nanoparticles (SeNPs) are considered as nontoxic and bioactive agents with low toxicity and high biocompatibility [5]. erefore, SeNPs are described as nanomaterials that could be used for therapeutic purposes. Besides, SeNPs as a strong antibacterial agent have been authenticated to inhibit bacterial growth [6]. Biofilms are complex communities of surface-bound bacteria that are embedded together in a selfproduced matrix. Since these communities of bacteria are difficult to treat, there is a need for novel antibiofilm inhibitors [7].
However, previous studies have not specifically evaluated the potential antimicrobial and antibiofilm activity of SeNPs against V. cholerae strains.
By relying on these results, the present study was performed to investigate the antimicrobial and antibiofilm effect of SeNPs on V. cholerae strains. erefore, SeNPs were first synthesized using a chemical reduction approach, then their antibacterial and antibiofilm activity against V. cholerae O1 ATCC 14035 strain was evaluated. e cytotoxic potential of SeNPs was evaluated against Caco-2 cell line. Herein, the antimicrobial and antibiofilm activity of SeNPs against V. cholerae O1 ATCC 14035 was reported for the first time. All of the findings strongly verified the potential antimicrobial and antibiofilm effect of SeNPs as a novel therapeutic agent against V. cholerae O1 ATCC 14035.

Synthesis of Selenium Nanoparticles (SeNPs).
SeNPs was synthesized using reducing sodium selenite in the presence of ascorbic acid according to a study by Vahdati slightly modified. Synthesis of SeNPs by this method has biocompatibility and good reducing properties [9,16]. Briefly, 58.13 mM ascorbic acid (Merck, Germany) was added to Na 2, SeO 3 (5H 2 O) at a concentration of 1.2 mM (Merck, Germany). Ascorbic acid was introduced into the resulting solution at a 4 : 1 ratio of ascorbic acid/Na 2 SeO 3 .
Ascorbic acid was added dropwise while stirring at 1300 rpm at room temperature. e formation of SeNPs was visible with a color change in the solution from white to orange. e solution was centrifuged at 12000 rpm and then pellet washed. Pellet was resuspended in 1 mL of sterile double-distilled water. In addition, Tween 20 (30 μL/20 mL) was used to prevent the aggregate of SeNPs during the synthesis process.

Characterization of SeNPs.
e prepared SeNPs identity was verified using an ultraviolet-visible (UV-Vis) spectrophotometer (Perkin-Elmer, ermo Scientific, USA) in the 200-500 nm wavelength range. e size distribution and zeta potential of the prepared SeNPs were specified using a Zeta Sizer Nano Series (Zetasizer Nano ZS, Malvern, Worcestershire, UK) after sonicating for 10 min in a bath-type sonicator.
Using the Inductively Coupled Plasma-Atomic Absorption Spectroscopy (ICP-AAS) technique, the amount of selenium in the prepared nanoparticles was determined. e concentration of SeNPs was determined according to the standard selenium concentration curve. Selenium standards were made from sodium selenite salt at concentrations of 0-100 ppm.
Acid digestion of nanoparticles was carried out using a solution of 2% nitric acid. Selenium standards were then prepared from sodium selenite salt at concentrations of 1-100 ppm. In order to identify the major structural groups in SeNPs, Fourier transform infrared (FTIR) analysis was performed using a FTIR spectrometer (FTIR, PerkinElmer, USA) in the wavenumber range of 400-4000 cm −1 .
In addition, both scanning electron microscopy (FE-SEM, Tescan Mira3) and transmission electron microscopy (TEM, Phillips EM 2085) (100 kV) methods were employed to study and analyze the morphology of the prepared nanoparticles. All reported images were estimated by ImageJ software.

Sterility Test.
In this study to investigation of sterility of prepared nanoparticles, SeNPs were cultured on the following media, including thioglycolate media, nutrient agar, blood agar, MacConkey agar, and Sabouraud dextrose agar, and then placed under anaerobic and aerobic conditions. e optimal culture period was also examined [17].
To obtain the best results, the cells were implanted in a 96well plate with approximately 10 6 cells per well in their exponential growth phase and inoculated with different concentrations (0-200 μg/mL) of SeNPs for 24, 48, and 72 h after incubation in CO 2 incubator. e supernatant of Caco-2 cells without stimuli was used as negative control. Each test was performed along with a control containing complete medium with no cells as blank; nanoparticles and MTT reagent without cells were used as blanks. Following exposure to the composite, 100 μL of reconstituted MTT was added to each well and incubated again for 4 h. Finally, detergent reagent with a volume equal to the volume of the original culture medium (usually 100 μL) was added up and down by a pipet to completely dissolve the resulting MTT formazan crystals. After thorough mixing, the resulting solution optical density was read immediately in a microplate reader using a microplate spectrophotometer (Epoch, USA) at the background absorbance of multiwell plates at 690 nm and subtract at 570 nm. All assays were carried out in triplicate. Cell viability was represented and compared to the controls. e viability of controls (without stimuli) was set at 100%, and all values were represented as percentage. Respective IC50 values (minimum particle concentration causing 50% cell death) were determined by GraphPad Prism Software Version 8 by employing regression analysis. V. cholerae strains were cultured in Mueller-Hinton broth (MHB) culture medium at 37°C for 24 h and grown to log phase to reach an optical density (OD) of 1 : 0 (10 8 CFU/mL). After 24 h, 5 × 10 5 CFU/mL (0.01 mL) of bacteria (V. cholerae O1 ATCC 14035) and 0.1 mL of MHB were added to each well and incubated overnight at 37°C for 24 h. Each sample was then serially diluted to obtain a dilution of 10 −5 . Culture medium containing bacteria without SeNPs was prepared as positive control, and culture medium without bacteria was prepared as negative control. To count the colonies, 10 μL of each sample was cultured onto Mueller-Hinton agar (MHA) and incubated at 37°C for 24 h. After incubation, CFU of bacterial cells was enumerated based on the following formula: number of colonies × 100 × inverse dilution factor (M07, CLSI, 2019).

Antibacterial Activity Evaluation
All experiments were performed with three replicates.

Antibiofilm
Assay. e antibiofilm activity of SeNPs was assessed in this study using crystal violet method. Briefly, V. cholerae O1 ATCC 14035 strain was cultured in 1 mL of BHI (brain-heart infusion) broth medium (Merck, Germany) at 37°C for 24 h to obtain an optical density (OD) of 1 : 0 (10 8 CFU/mL). Each well of a 96-well plate was filled with 0.1 mL of BHI broth supplemented with 0.5% (w/v) sucrose. e wells were then inoculated with 0.1 mL of SeNPs (concentrations used were 0-200 mg/mL) and 0.01 mL of V. cholerae O1 ATCC 14035 suspension (10 8 CFU/mL) and incubated at 37°C for 24 h. e medium of each well was gently taken away, and the wells were rinsed three times with 0.2 mL of phosphate-buffered saline (PBS PH7.2) to eliminate free-floating bacterial strains. Attachment of V. cholerae O1 ATCC 14035 to the 96-well plate was evaluated by staining with 1% (wt/vol) crystal violet solution.
e 96-well plate was again washed to remove excess stain and kept for drying. Finally, the biofilm mass was destained using 95% ethanol for 45 min, and a microplate spectrophotometer (Epoch, USA) was used to measure the OD value of biofilm formation related to crystal violet at 570 nm. e optical density (OD) values of different samples with SeNPs were compared with the control sample.
e OD value was considered as biofilm formation on the surface of the 96-well plate. e test was performed in triplicate [18].

Statistical Analysis.
To assess the significant differences, GraphPad Prism Software Ver. 8 was used by employing one-way ANOVA test. A P value of less than 0.05 was considered as significant. e obtained results were represented as the mean ± standard deviation (SD). All experiments were performed in triplicate.

Characterization of SeNPs.
e appearance of a sharp peak at around 266 nm displayed by UV-Vis absorption spectra is assigned to SeNPs. e characterization of SeNPs absorption peak is shown in Figure 1.
Also, the color change from white to orange shows a decrease in selenium to elemental selenium (Se 0 ) and the formation of Se nanoparticles shown in Figure 2.
According to the DLS (dynamic light scattering) analysis results, SeNPs zeta potential was determined to be about −32.2 mV. e average size of the synthesized SeNPs was determined to be around 71.1 ± 10.3 nm. e polydispersity index (PDI) in DLS analysis was 0.21, confirming the homogeneous and nondispersive size of SeNPs (Figure 3).
Using ICP method, the calibration curves were plotted for selenium standards, and selenium content in nanoparticles was determined to be 0.654 μg/ml (Figure 4).
Furthermore, Se-OH groups and nanoparticle surface hydroxyl groups also peak in the range of 400-4000 cm −1 .
e surface groups of Se-C have an absorption peak in the range of 1000-1800 cm −1 due to the presence of ascorbic acid in the synthesis of these particles. ese functional groups confirm the contribution of different reducing and stabilizing agents in the synthesis of SeNPs [19][20][21]. FTIR spectra of SeNPs are shown in Figure 5. e absorption 1221.62 cm −1 represents that the complexation takes place between C-N or -C-N group and selenium ions [21]. e line at 1321 cm −1 after the synthesis of SeNPs was attributed to C-H band vibrations, or syringyl ring breathing with C�O stretching. e presence of a peak Canadian Journal of Infectious Diseases and Medical Microbiology at 758 cm −1 was assigned to the ring-vibrating modes of ortho-substituted aromatics [22]. Bands detected at 820, 1024, and 1321 are related with C-O stretch [20,23]. e band at 1677 cm −1 might represent the asymmetric and symmetric stretching vibrations of carboxylates (some carboxylic acids as products of deep ascorbic acid oxidation adsorbed on SeNPs) [24]. In the FTIR spectrum of SeNP, the peaks in the range 3200-3526 cm −1 and 820 cm −1 signifying correspond to the diverse hydroxyl groups (-OH). A peak at 3415 cm −1 is assigned to O-H stretching vibration of alcohol and phenol groups. e peak at 3513.38 cm −1 corresponds to the bending vibration of O-H. e bands at 628 cm −1 correspond to the stretching and bending vibrations of Se-O, which may be attributed to the binding of SeNPs to the carbonyl groups from the yields of oxidation of ascorbic acid [24,25]. e morphology and size distribution of selenium nanoparticles were investigated using FE-SEM and TEM, showing approximately a spherical and regular shape with an average size of 22.6 nm, the results of which are presented in Figure 6.

Sterility Test.
In this test, SeNPs were cultured on the thioglycolate media, nutrient agar, blood agar, and Mac-Conkey agar and then placed under anaerobic and aerobic  conditions. Examination of bacterial cultures after 24 and 48 h showed no bacterial growth in the media, revealing the sterility of the synthetized SeNPs.

Cytotoxic Effect of SeNPs.
In this study, the cytotoxic potential of SeNPs (after sterilization) was evaluated against Caco-2 cells using MTT test; the results were determinate as the percentage of cell viability in the presence of different concentrations of SeNPs (Figure 7). e viability of controls (without stimuli) was set at 100%. According to the results, more than 50% of the cells treated with 0-200 ppm of SeNPs for 24 and 48 h were viable. Moreover, the cell viability in the presence of 100 μg/mL of SeNPs after 24 and 48 h was 83.1 and 78.8%, respectively. Figures 8 and 9, SeNPs significantly inhibited bacterial growth. e antibacterial effect of different concentrations of SeNPs was evaluated separately. According to the results, the antibacterial effect of SeNPs was more evident at higher concentrations. In fact, the bacterial growth was inhibited in the presence of SeNPs at concentrations higher than 25 μg/mL. e bacterial growth was completely inhibited in cells treated with 200 and 100 μg/mL of SeNPs. Bacterial growth was reduced in the presence of SeNPs at concentrations of 50 and 25 μg/mL. According to the results, the antibacterial activity of SeNPs was significant at concentrations of 200, 100, 50, 25, and 12.5 μg/mL ( * * * * P value <0.0001) compared to the positive control.

Antibiofilm Assay.
e antibiofilm activity of SeNPs was evaluated against V. cholerae O1 ATCC 14035 strain using crystal violet assay, the results of which are presented in Table 1 and Figure 10. e antibiofilm activity of SeNPs was higher at concentrations of 200, 100, and 50 μg/mL ( * * * * P value <0.0001) compared to the concentrations of 25 ( * * * P value <0.001) and 12.5 μg/mL ( * P value <0.05). Biofilm formation was determined as nonadherent at concentrations of 200 and 100, as weak at a concentration of 50, and as strong at concentrations of 25, 12.5, and 0; however, biofilm formation in no group was determined as intermediate. e results exhibited that SeNPs had inhibitory activity against biofilm formation at concentrations higher than 50 μg/ml. Biofilm formation was measured photometrically at OD � 570 nm, the results of which are presented in Figure 11 [16,26], reporting the nontoxic nature of SeNPs (100 μg/mL). In other study, the concentration 41.5 ± 0.9 μg/mL of SeNPs had 50% cell death (IC50) in the cells treated [27] . is result is in agreement with a previous study that shows Se NPs exhibit a mild cytotoxic activity on Caco-2 cells [28]. Indumathy et al.
Previous studies have suggested that nanoparticles could be used as antibacterial agents against V. cholerae [30][31][32].
is study results suggest that SeNPs could be considered as an effective antibacterial and antibiofilm agent and utilized as an efficient approach against V. cholerae infections. SeNPs were synthesized in this study by reducing sodium selenite in the presence of ascorbic acid. SeNPs were produced with a diameter of around 71.1 ± 10.3 nm. e present study results showed that SeNPs (12.5-200  e results demonstrated that SeNPs (spherical in shape with an average diameter of ∼79 nm) could be potentially employed as an antibacterial agent for inhibiting S. aureus growth as well as for food safety applications at the concentration 20-50 μg/mL [28].
In a research carried out by Guisbiers et al., antibacterial activity of SeNPs was assessed against E. coli and S. aureus isolates. eir study results showed that SeNPs significantly diminished the count of E. coli and S. aureus strains after 4, 8, and 24 h [8]. e mechanism of antibacterial action of SeNPs is unknown. Research has shown that SeNPs could increase the lag time and substantially reduce the growth rate of S. aureus through depleting glutathione (GSH) [33].
In a study by Zhang et al., SeNPs exhibited inhibitory activity against bacterial growth, and the mortality rate of Gram-negative bacteria was much better than that of other bacteria. SeNPs caused the leakage of proteins and polysaccharides after reacting with bio-SeNPs by altering membrane permeability and disrupting bacterial cell walls. Also, changes in the intensity of reactive oxygen species (ROS) by SeNPs could induce antibacterial effects [34].
e results of antibacterial ability of some studies were shown in Table 2.
e results of different studies vary in antibacterial effects of SeNPs on bacteria.
e difference between the findings of different studies is mainly due to the difference in the size of nanoparticles and the type of bacteria used. One of the most important factors affecting the antimicrobial properties of nanoparticles is the particle size and concentration. It was considered that smaller nanoparticles had increased the production of ROS than larger surface area to volume ratio inside or out of the cells [38].
In this study with the increase of SeNPs concentration (12.5 to 200 μg/mL), the growth of V. cholerae gradually reduced ( Figure 8). e results significantly indicated that SeNPs have potential antibacterial activity against V. cholerae O1 ATCC and could be employed as an adjunctive antibacterial treatment for V. cholerae infections.
New strategies other than conventional antibiotic treatments are needed to control biofilm formation in bacterial infections. V. cholerae biofilms have been shown to be hyperinfective; these strains could remain in the environment and increase antibiotic resistance in V. cholerae strains. Mature biofilm formation requires the production of matrix proteins, especially RbmA, RbmC, and Bap1. ese proteins maintain the structural integrity of the wild-type biofilm [39].
Various studies have shown the effect of different nanoparticles as biofilm inhibitors against different bacteria. e antibiofilm activity of magnesium oxide against Streptococcus mutans and Streptococcus sobrinus (500 μg/mL) was indicated by Noori and Kareen. ey reported that the average nanoparticle is approximately 20.8 nm. [40]. e synthesized AgNPs (the spherical shape, at the size of 55 nM) were used as an inhibitor for controlling biofilm  formation against Klebsiella pneumoniae. AgNP concentration of 100 μg/ml was evaluated through the percentage biofilm inhibition 64% for K. pneumoniae strain MF953600 and 86% for MF953599 [41]. e results are consistent with the findings of a previous study by Shakibaie et al. [13], reporting that SeNPs at the concentration of (0-16 μg mL −1 ) therapy inhibited the biofilm formation of S. aureus, P. aeruginosa, and P. mirabilis by 42, 34.3, and 53.4%, respectively. In their study, SeNPs was made with spherical shape and diameter range of 80 to 220 nm synthesized by Bacillus sp. [12]. Contrary to this study results, Haney demonstrated that iron oxide nanoparticles at a concentration of 0.2 mg/mL could increase biofilm biomass [42]. e results showed that the potential of SeNPs as an inhibitor of biofilm formation against V. cholerae was described ( Figure 9).
In short, this study showed that SeNPs could be used as an antibiofilm and antibacterial agent against V. cholerae infections. e limitation of our research included the lack of an in vivo study, which is intended to be carried out in future efforts.

Conclusion
e synthesized nanostructures exhibited high antibiofilm and antibacterial activity against V. cholerae, which is considered as a major public health concern. In conclusion, SeNPs could be considered as a new therapeutic nanostructure with high antibacterial and antibiofilm potential and used as a promising alternative for V. cholerae infections therapy in clinical settings. erefore, SeNP might be useful for various pharmaceutical applications.
Data Availability e datasets used and/or analyzed during the current work are available from the corresponding authors on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interests.

Authors' Contributions
SBJ performed the experiments and drafted the manuscript. BB developed and supervised the work. SS and BB contributed to data interpretation. All authors reviewed the manuscript. All authors read and approved the final manuscript (Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Jalal-Ale-Ahmad Ave, Tehran 14117-13116, Iran).